The Maize Divergent spindle-1 (dv1) Gene Encodes a Kinesin-14A Motor Protein Required for Meiotic Spindle Pole Organization

Abstract

The classic maize mutant divergent spindle-1 (dv1) causes failures in meiotic spindle assembly and a decrease in pollen viability. By analyzing two independent dv1 alleles we demonstrate that this phenotype is caused by mutations in a member of the kinesin-14A subfamily, a class of C-terminal, minus-end directed microtubule motors. Further analysis demonstrates that defects in early spindle assembly are rare, but that later stages of spindle organization promoting the formation of finely focused spindle poles are strongly dependent on Dv1. Anaphase is error-prone in dv1 lines but not severely so, and the majority of cells show normal chromosome segregation. Live-cell imaging of wild type and mutant plants carrying CFP-tagged β-tubulin confirm that meiosis in dv1 lines fails primarily at the pole-sharpening phase of spindle assembly. These data indicate that plant kinesin-14A proteins help to enforce bipolarity by focusing spindle poles and that this stage of spindle assembly is not required for transition through the spindle checkpoint but improves the accuracy of chromosome segregation.

Keywords: kinesin-14A; maize; meiosis; mutant; spindle; tubulin.

Figure 1

Two alleles of dv1 show a divergent spindle phenotype. Meiocytes were stained using immunofluorescence with a primary antibody specific to α-tubulin. Scale bars for each image represent 10 μm. (A) Wild type spindle showing highly focused spindle poles; (B) dv1-1/dv1-1 spindle showing splayed, divergent poles; (C) Expression of the dv1-1 allele of ZmKin6 adjusted to the B73 wild type allele. Error bars represent 95% confidence intervals, groups of significant difference are designated with lowercase letters; (D) dv1-1/dv1-IG heteroallelic mutant is similar to the dv1-1 homozygote; (E) Gene model of ZmKin6 highlighting the location of the two dv1 alleles, the dv1-1 stop codon in the sixth exon and the dv1-IG transversion in the motor domain. Coordinates along chromosome 2 are shown above. The locations of sequencing primers are shown with red arrows while the locations of genotyping primers are shown with blue arrows. The reference and mutant sequences of each allele are shown below the gene diagram.

Figure 2

Quantification of the dv1-1 phenotype on spindle shape and pollen viability. Spindles were visualized using immunofluorescence as shown in Figure 1. Measurements were taken using the Slidebook 6 digital microscopy software package (Intelligent Imaging Innovations, Denver, CO). Lowercase letters in B, C, and D represent groups of significant difference as determined using ANOVA at α = 0.05. (A) Schematic of measurements taken, including width of the metaphase plate (W_C), length of the half-spindle from metaphase plate to pole (L), and width of the spindle at 75% of length L (W_S). The ratio of W_S/W_C can be used to quantify spindle shape as either focused (shown in the top half-spindle) or divergent (bottom half-spindle); (B) Width of the metaphase plate (μm) is significantly higher in plants homozygous for dv1-1 than either wild type or heterozygous plants; (C) Length of the half-spindle (μm) is significantly larger than wild type in both dv1-1 homozygotes and heterozygotes; (D) The ratio of spindle width at the metaphase plate to spindle width near the poles, a proxy for spindle shape and degree of pole focus, is significantly higher in dv1-1 plants while heterozygotes displayed an intermediate phenotype.

Figure 3

Rarely-observed severe effects of dv1-1 include multiple spindles and multinucleate daughter cells. Meiocytes were stained using immunofluorescence with a primary antibody specific to α-tubulin shown in red and CENP-C shown in green. DNA (DAPI) is shown in blue. Scale bars for each image represent 10 μm. (A) Multi-spindle dv1-1 cell with metaphase alignment errors. Poor congression of chromosomes along the metaphase plate in a dv1-1 cell with parallel spindles in metaphase separated by space (arrows); (B) Early anaphase of a similar dv1-1 cell with parallel spindles and chromosomes separated by space (arrow); (C) Cells with severe errors in spindle assembly. Cell on the left appears to be a fusion of a small and a large spindle in the process of correction. Cell on the right shows two distinct spindles, the larger of which appears as a chaotic array of early prometaphase; (D) Two separate spindles of approximately the same size in a dv1-IG cell; (E) Tri-polar spindle of a dv1-1 cell at metaphase showing separated microtubules (arrow) with congressed chromosomes; (F) Lagging chromosomes during anaphase in a dv1-1 cell; (G) Tetrad-stage cells showing examples of mininuclei (arrows), isolated chromosomes not a part of the nucleus; (H) Pollen shows a decreased viability in both dv1-1 homozygotes and heterozygotes. Groups of statistical significance are designated with lowercase letters. The heterozygous phenotype was not statistically significant from either wild type or dv1-1 homozygous. (n = 7–10 plants per genotype, 500 pollen grains per plant; α = 0.05).

Figure 4

Live cell imaging demonstrates a role for Dv1 in prometaphase and metaphase. Images captured from a line carrying a β-tubulin transgene tagged with a cyan fluorescent protein (shown in white) and incubated with SYTO12 which stains chromosomes (shown in blue). Images presented are sequential frames from a single movie with minutes since the original capture shown in the lower right of each image. Scale bars for each image represent 10 μm. Movies of all cells can be found in Movie S1. (A) Chromosomes in a wild type cell begin loosely collected in a metaphase plate and then compress as the spindle narrows throughout metaphase; (B) Chromosomes in a dv1-1 cell are highly unorganized in prometaphase with three separate mini-spindles around different chromosome groups which are then brought together; (C) The spindle poles of a wild type cell begin rounded, then sharpen and elongate before the cell enters anaphase; (D) The spindle pole of a heterozygous cell shows highly focused spindles which curl along the edge of the cell as it enters anaphase; (E) The spindle pole of a dv1-1 cell is highly unorganized in metaphase and anaphase chromosome movement is uneven with several lagging chromosomes; (F) Measurement of chromosome offset, the distance between the center of the chromosome mass and the spindle appears to be larger in the dv1-1 homozygote than other genotypes; (G) Distance of chromosomes moved in anaphase A appears to be larger in the dv1-1 heterozygotes and homozygotes; (H) Rate of chromosome movement in anaphase A is not significantly different between the three genotypes.